Varicella Zoster Virus infects mucosal associated Invariant T cells

Abstract

Introduction: Mucosal Associated Invariant T (MAIT) cells are innate-like T cells that respond to conserved pathogen-derived vitamin B metabolites presented by the MHC class I related-1 molecule (MR1) antigen presentation pathway. Whilst viruses do not synthesize these metabolites, we have reported that varicella zoster virus (VZV) profoundly suppresses MR1 expression, implicating this virus in manipulation of the MR1:MAIT cell axis. During primary infection, the lymphotropism of VZV is likely to be instrumental in hematogenous dissemination of virus to gain access to cutaneous sites where it clinically manifests as varicella (chickenpox). However, MAIT cells, which are found in the blood and at mucosal and other organ sites, have yet to be examined in the context of VZV infection. The goal of this study was to examine any direct impact of VZV on MAIT cells.

Methods: Using flow cytometry, we interrogated whether primary blood derived MAIT cells are permissive to infection by VZV whilst further analysing differential levels of infection between various MAIT cell subpopulations. Changes in cell surface extravasation, skin homing, activation and proliferation markers after VZV infection of MAIT cells was also assessed via flow cytometry. Finally the capacity of MAIT cells to transfer infectious virus was tested through an infectious center assay and imaged via fluorescence microscopy.

Results: We identify primary blood-derived MAIT cells as being permissive to VZV infection. A consequence of VZV infection of MAIT cells was their capacity to transfer infectious virus to other permissive cells, consistent with MAIT cells supporting productive infection. When subgrouping MAIT cells by their co- expression of a variety cell surface markers, there was a higher proportion of VZV infected MAIT cells co-expressing CD4+ and CD4+/CD8+ MAIT cells compared to the more phenotypically dominant CD8+ MAIT cells, whereas infection was not associated with differences in co-expression of CD56 (MAIT cell subset with enhanced responsiveness to innate cytokine stimulation), CD27 (co-stimulatory) or PD-1 (immune checkpoint). Infected MAIT cells retained high expression of CCR2, CCR5, CCR6, CLA and CCR4, indicating a potentially intact capacity for transendothelial migration, extravasation and trafficking to skin sites. Infected MAIT cells also displayed increased expression of CD69 (early activation) and CD71 (proliferation) markers.

Discussion: These data identify MAIT cells as being permissive to VZV infection and identify impacts of such infection on co- expressed functional markers.

Keywords: MAIT cells; Varicella Zoster Virus; herpesvirus; innate-like T cells; productive infection.

Figure 1

VZV infects MAIT cells from human peripheral blood. Human PBMCs were inoculated with mock or clinical VZV isolate (VZV-S) infected ARPE-19 epithelial cells for 2 days and then analyzed for infection by flow cytometry. (A) Representative flow cytometry plots depicting gating strategy of CD3⁺ MR1-Tetramer⁺ (MR1-Tet⁺) MAIT cells, non-MAIT (ie MR1-Tet^-) CD3⁺ CD4⁺ (CD4 T cells) and non-MAIT (ie MR1-Tet^-) CD3⁺ CD8⁺ cells (CD8 T cells), as well as quantifying surface VZV glycoprotein (g)E:gI expression on gated populations. (B) Frequencies of total live gE:gI⁺ lymphocytes (shaded), compared to MAIT cells, non-MAIT CD4⁺ and non-MAIT CD8⁺ cells (n=14). Symbols represent individual donors across the lymphocyte populations, with mean and standard error of mean (SEM) indicated by the bars. Statistical analysis between gE:gI expression on specific lymphocyte populations was performed via repeated measures (RM) one-way ANOVA with the Greenhouse-Geisser correction and Tukey’s multiple comparisons test.

Figure 2

VZV infects diverse MAIT cell subsets. Human PBMCs were inoculated with mock or VZV-S infected ARPE-19 epithelial cells for 2 days and then analyzed for infection by flow cytometry as per surface VZV-gE:gI expression (A) Graphs showing frequencies of various MAIT cell subpopulations (n=4-14). (B) Frequencies of gE:gI⁺ lymphocytes within each subpopulation depicted, with symbols representing individual donors across the MAIT subpopulations, with mean and SEM indicated by the bars. Statistical analysis of VZV gE:gI expression, comparing CD4⁺ cells with CD8⁺ cells, and CD4⁺/CD8⁺ cells compared to CD4^-/CD8^- cells was performed via RM one-way ANOVA with the Greenhouse-Geisser correction and Tukey’s multiple comparisons test (n=14).***p<0.001. Statistical analysis of gE:gI expression on MAIT cells expressing CD56^- was compared to those expressing CD56⁺ (n=14), CD27^- compared to CD27⁺ (n=4) and PD-1^- compared to PD-1⁺ (n=4) was performed via two tailed paired t test.

Figure 3

VZV infection of MAIT cells is associated with expression of early activation and proliferation markers. Human PBMCs were inoculated with mock or VZV-S infected ARPE-19 epithelial cells for 2 days and then analyzed for infection (gE:gI), proliferation (CD71), and early activation (CD69) markers by flow cytometry. (A) Graph shows comparative frequency of surface CD71 and CD69 expression in mock inoculated non-MAIT (ie MR1-Tet^-) CD3⁺ T cells and MAIT cell populations, with symbols representing individual donors (n=6). Statistical analysis was performed via two tailed paired t test. **p<0.001, ***p<0.001. (B) Representative histograms show expression of CD71 and CD69 by MAIT cells for Mock (blue) and VZV infected (VZV⁺) (red) populations corresponding to their respective isotype controls (filled grey). Flow cytometry plots show surface expression of CD71 and CD69 on MAIT cells for Mock (blue) and VZV infected (VZV⁺) (red) populations. Graphs show frequency of CD71 and CD69 in non-MAIT (ie MR1-Tet^-) CD3⁺ and MAIT cell subpopulations, with symbols representing individual donors, and mean and SEM indicated by the bars. Statistical analysis of CD71 and CD69 expression between Mock and VZV⁺ infected non-MAIT CD3⁺ cells and MAIT cells was performed via two tailed paired t test (n=6). *p<0.05, ****p<0.0001. (C) Flow cytometry plots show CD69 vs CD71 double expression on Mock (blue) and VZV infected (VZV⁺) (red) populations. Graph shows frequency of CD69/CD71 double expressing MAIT cells, with symbols representing individual donors, and mean and SEM indicated by the bars. Statistical analysis of CD71 and CD69 expression between Mock and VZV⁺ infected non-MAIT CD3⁺ cells and MAIT cells was performed via two tailed paired t test (n=6). *p<0.05, ****p<0.0001.

Figure 4

VZV infection of MAIT cells does not suppress CCR2, CCR5 and CCR6 expression. Human PBMCs were inoculated with mock or VZV-S infected ARPE-19 epithelial cells for 2 days and then analyzed for infection (gE:gI), and extravasation chemokine receptor markers (CCR2, CCR5 and CCR6) by flow cytometry. (A) Graph shows comparative frequency of surface CCR2, CCR5, and CCR6 expression in mock inoculated non-MAIT (ie MR1-Tet^-) CD3⁺ T cells and MAIT cell populations, with symbols representing individual donors (n=4). Statistical analysis was performed via two tailed paired t test. **p<0.001, ***p<0.001. (B) Representative histograms show expression of CCR2, CCR5 and CCR6 by MAIT cells for Mock (blue) and VZV infected (VZV⁺) (red) populations corresponding to their respective isotype controls (filled grey). Flow cytometry plots show surface expression of CCR2, CCR5 and CCR6 on mock infected (blue) and VZV infected (red) MAIT cell populations. Graphs show frequencies of non-MAIT CD3⁺ T cells and MAIT cells expressing CCR2, CCR5 and CCR6, with symbols representing individual donors, with mean and SEM indicated by the bars. Statistical analysis of CCR2, CCR5 and CCR6 expression between Mock and VZV⁺ infected non-MAIT CD3⁺ cells and MAIT cells was performed via two tailed paired t test (n=4). *p<0.05.

Figure 5

VZV infection of MAIT cells retains expression of CLA and CCR4 skin homing chemokine receptor expression. Human PBMCs were inoculated with mock or VZV-S infected ARPE-19 epithelial cells for 2 days and then analyzed for infection (gE:gI), and skin homing markers (CLA and CCR4) by flow cytometry. (A) Graph shows comparative frequency of surface CLA, and CCR4 expression in mock inoculated non-MAIT (ie MR1-Tet-) CD3⁺ T cells and MAIT cell populations, with symbols representing individual donors (n=5). Statistical analysis was performed via two tailed paired t test. ***p<0.001. (B) Representative histograms show expression of CLA and CCR4 by MAIT cells for Mock (blue) and VZV infected (VZV⁺) (red) populations corresponding to their respective isotype controls (filled grey). Flow cytometry plots from one donor show surface expression of CLA and CCR4 on MAIT cells for Mock (blue) and VZV infected (VZV⁺) (red) populations. Graphs show frequencies of non-MAIT CD3⁺ and MAIT cells expressing CLA and CCR4 with symbols representing individual donors across the subpopulations, with mean and SEM indicated by the bars. Statistical analysis of CLA and CCR4 expression between Mock and VZV⁺ infected non-MAIT CD3⁺ and MAIT cells was performed via two tailed paired t test (n=5). *p<0.05, ***p<0.001.

Figure 6

MAIT cells support de-novo viral replication and virus transmission. Human PBMCs were inoculated with mock or a GFP-tagged VZV infected ARPE-19 epithelial cells for 2 days and then FACS sorted for MAIT cells (CD3⁺ MR1-Tetramer⁺ Vα7.2⁺). (A) Representative flow cytometry plot depicts MAIT cell frequencies of total live lymphocytes pre- and post-sorting. (B, C) Sorted mock or VZV-GFP exposed MAIT cells were citrate buffer washed three times and then added to ARPE-19 epithelial cell monolayers at a ratio of 1 sorted MAIT cell to 5 epithelial cells. Co-cultures were incubated at 37°C 5% CO₂ and five days later monolayers were fixed, counterstained with DAPI and infectious centers visualized by the detection of VZV-GFP by fluorescent microscopy. Representative images (from four replicate experiments) of ARPE-19 monolayers exposed to (B) mock infected MAIT cells or (C), inset in (D) VZV-GFP exposed MAIT cells are shown.